Biochemistry and genetics of the ORMDL regulators of sphingolipid biosynthesis

鞘脂生物合成 ORMDL 调节因子的生物化学和遗传学

• 批准号: 9323552
• 负责人: BRIAN W. WATTENBERG
• 金额: $ 38.13万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9323552
• 关键词: Address; Affect; Agonist; Alpha Cell; Anabolism; Architecture; Asthma; Binding; Binding Sites; Biochemistry; CRISPR/Cas technology; Cardiovascular Diseases; Cell Line; Cell membrane; Cell-Free System; Cells; Ceramides; Childhood Asthma; Complex; Conserved Sequence; Detergents; Disease; Elements; Endoplasmic Reticulum; Enzymes; Epithelial; Genes; Genetic; Goals; Homologous Gene; Individual; Inflammatory; Knowledge; Lipids; Malignant Neoplasms; Measures; Mediating; Membrane; Membrane Proteins; Metabolic; Metabolism; Molecular; Mutation; Pathway interactions; Phenotype; Population Study; Protein Isoforms; Regulation; Research; Risk; Signal Transduction; Single Nucleotide Polymorphism; Smooth Muscle Myocytes; Specificity; Sphingolipids; Stimulus; Suggestion; System; Technology; Testing; Yeasts; asthmatic; cell type; eosinophil; genome editing; genome wide association study; novel; physical property; reconstitution; response; risk variant; serine palmitoyltransferase; tool

Sphingolipids are a diverse but metabolically related group of lipids with unique functional and physical properties that are critical for cell signaling and membrane architecture. Misregulation of particular sphingolipids has been implicated in such diverse diseases as cancer, inflammatory disease, and cardiovascular disease. In particular, as outlined below, genome-wide association studies have strongly implicated the subject of this proposal, the sphingolipid metabolic regulator ORMDL, in the risk for childhood asthma. The overall level of cellular sphingolipid is determined by the initiating, and rate limiting enzyme in the biosynthetic pathway, serine palmitoyltransferase (SPT). Not surprisingly, SPT activity is homeostatically controlled-i.e. SPT activity is strongly inhibited by elevated levels of cellular sphingolipid. We have demonstrated that this homeostatic control is mediated by a set of small membrane proteins found in the endoplasmic reticulum, the ORMDLs. The goal of the studies proposed here is to establish the mechanism by which the ORMDLs mediate SPT inhibition in response to elevated sphingolipid levels and to begin to uncover how those mechanisms impact on risk for asthma. These studies will answer basic questions concerning how ORMDLs sense cellular sphingolipid levels and how changes in sphingolipid levels are transmitted through ORMDL to affect SPT activity. We will test the hypothesis that ceramide directly binds ORMDL to trigger SPT regulation. We will determine the structural elements in ORMDL that are essential for mediating sphingolipid regulation of SPT, will use a recently constructed cell-free system to quantitate the levels of sphingolipid sensed by the ORMDL system, and will test the hypothesis that elevation of sphingolipid induces changes in ORMDL topology. Finally we will construct a reconstituted system with purified ORMDL and SPT to test the hypothesis that the ORMDLs and SPT are the only required components of this regulatory system. In addition we propose to address an essential question concerning the genetics of ORMDL expression that has emerged from genome-wide association studies in asthma. Single nucleotide polymorphisms (SNPs) adjacent to the gene of one of the ORMDL isoforms, ORMDL3, are strongly and consistently associated with the risk for childhood asthma and with elevated expression of ORMDL3. Although the population-based studies are intriguing and suggestive, a detailed examination of this phenomenon has been hamstrung by the inability to directly test the effect of these SNPs on ORMDL3 expression. It has not been possible to directly measure the degree to which these SNPs affect ORMDL3 expression nor in what cell types and conditions this elevated expression occurs. Here we propose to use emerging Crispr/Cas9 genome editing technology to produce paired cell lines, in asthma-relevant cell types, containing either a risk or non-risk genotype at the most prominent risk-associated SNP. These will be used to examine the extent, cell type, and stimuli specificity of elevation of ORMDL3 expression induced by the risk allele and how this affects sphingolipid metabolism.

鞘脂是一类具有独特功能和生理功能的多种代谢相关的脂类。 对细胞信号和膜结构至关重要的特性。对特定项目的不当监管 鞘磷脂与多种疾病有关，如癌症、炎症性疾病和 心血管疾病。特别是，如下所述，全基因组关联研究具有很强的 这项提议的主题，鞘磷脂代谢调节剂ORMDL，与儿童的风险有关 哮喘。细胞鞘磷脂的总体水平由启动和限速酶决定。 生物合成途径，丝氨酸棕榈酰转移酶(SPT)。不足为奇的是，SPT的活动是内稳态的 受控--即SPT活性受到细胞鞘脂水平升高的强烈抑制。我们有 证明了这种动态平衡控制是由一组小的膜蛋白介导的，这些膜蛋白在 内质网，即ORMDLS。这里提出的研究的目标是通过以下方式建立该机制 ORMDL介导SPT抑制，以响应鞘磷脂水平的升高并开始揭示 这些机制如何影响哮喘的风险。这些研究将回答有关如何 ORMDL感知细胞鞘脂水平，以及鞘脂水平的变化是如何通过 ORMDL影响SPT活性。我们将检验神经酰胺直接与ORMDL结合以触发SPT的假设 监管。我们将确定ORMDL中对调节鞘磷脂至关重要的结构元素 SPT的调节，将使用最近构建的无细胞系统来量化鞘磷脂的水平 被ORMDL系统感知，并将检验神经鞘脂脂升高导致 ORMDL拓扑。最后，我们将用纯化的ORMDL和SPT构建一个重组系统来测试 假设ORMDL和SPT是该监管系统唯一必需的组件。此外 我们建议解决已经出现的关于ORMDL表达的遗传学的基本问题 来自对哮喘的全基因组关联研究。邻近的单核苷酸多态(SNPs) ORMDL亚型中的一种，ORMDL3的基因与高血压的风险密切相关。 儿童哮喘和ORMDL3表达升高。尽管基于人口的研究是 耐人寻味和具有启发性的是，对这一现象的详细研究因无法 直接检测这些SNP对ORMDL3基因表达的影响。还不可能直接测量 这些SNP对ORMDL3表达的影响程度，以及在什么细胞类型和条件下这种影响升高 出现了表达式。在这里，我们建议使用新兴的Crispr/Cas9基因组编辑技术来生产 哮喘相关细胞类型中的配对细胞系，最多包含风险或非风险基因 突出的与风险相关的SNP。这些将被用来检查范围、细胞类型和刺激的特异性 风险等位基因诱导的ORMDL3表达的升高以及这如何影响鞘磷脂代谢。